The invention resides in a process for determining the number of components in peaks, bands, and signals of chromatograms, electrograms, and spectrograms of all types obtained for analysis and substance separation wherever energy-correlated measurement values as a function of an evolving parameter appear and again disappear.
Modern analysis and separation techniques for any type of material proceed more and more toward a full automatization of the procedure from the sampling up to providing the desired final results. In such a procedure, the process scheme is repeated identically independently of the type of analysis. This is true generally for the locating and the identification of the material, but also for the sequencing of proteins, nucleic acids, carbohydrates, lipids, etc.: Progressive attachment, distribution on the basis of individual travel behavior and desorption of substances on various carrier materials and the subsequent substrate detection by way of dispositives sensitive thereto and preferably having a spectroscopic nature.
Experience has shown that, in addition to the homogeneous detection peaks which are generated by a single pure component (=100% peak purity), in other signals the spectral contributions of two or several components are mixed together (=mix-peaks).
The main reasons herefor are:
Quasi-identity of the components to be separated, for example, families of effective substances occur in the natural material chemistry. The individual components differ only slightly from one another.
The use of an unadapted separation process, which permits several components to appear in the same detection zone.
Peak determination under less than optimal conditions for the detection.
The peak purity and, if applicable, the number of spectrally overlapping components could not be easily recognized and utilized so far apparatus-logistically. When such uncertainties occur, the reaction of the apparatus is delayed and the final results are doubtful.
Highly developed measuring and separating processes, which are optimized in any other way, are limited in their efficiency by this general uncertainty factor.
U.S. Pat. No. 5,596,135 A discloses a process, which is based on the extinction difference diagrams. With this process, it can be determined whether a peak in a chromatogram is caused by one or several components. If there is more than one component, the number of components cannot be clearly determined.
EP 0 486 030 A discloses a similar method, which is based, however, on extinction diagrams.
Furthermore, EP 0 294 121 A discloses a method, wherein the number of components of a mixture can be determined using a mathematically complicated main component analysis (on the basis of a matrix with the determination of individual values and vectors).
It is the object of the present invention to provide a simple method by which the number of components in a mixture can be clearly determined.
In a process for determining the number of components involved in the formation of peaks, bands, and signals which are obtained in spectrograms where energy-correlated measurement values, such as extinctions, increase and decrease as a function of an evolving parameter, such as time, four different measurement values with at least three evolving parameter values are determined, three differences are formed from the four energy-correlated measurement values on the basis of the same evolving parameter, two quotients are formed from the respective differences and the two quotients are plotted over one another in a diagram, whereby a point is obtained if one component is responsible, a straight line is formed if two components are responsible, and a curve is formed if more than two components are responsible for the formation of the peaks, bands, or signals.
The method permits a precise and rapid determination of the number of components in peaks, bands, and signals, which are obtained for the analysis and component separation in all kinds of spectrograms, where energy correlated measurement values increase decrease as a function of an evolving parameter. The plurality of measurement values are derived from a particular such parameter value and differently energy-correlated so as to form characteristic geometric figures, in which the original evolving parameter is not contained. The figures obtained are reduced, depending on their complexity, in steps, first to straight lines and then to points. In this way, the number of the components involved in the formation of the peaks and consequently the composition of the whole spectrogram and also of the component mixture to be examined and/or separated is obtained in a simple manner.
In a preferred embodiment of the invention, instead of the energy-correlated direct-measurement values, the differences thereof or quotients thereof or even the quotients of the differences are placed in relation to one another in a simple way or in multiple ways. It becomes progressively possible thereby to extract 2, 3, 4 or more individual components from the peak and to identify and separate them. Also, the areas in a particular parameter range surrounded by the respective energy measurement values may be placed in relation to each other as integrals, whereby geometric figures are provided from which the peak compositions and analysis results can be obtained. This additionally improves the signal/noise ratio with a corresponding gain in sensitivity of the respective technical method.
A variant of the process for determining the number of components of spectrograms, wherein the structures are energy-correlated measurement values, which appear and disappear as a function of an evolving parameter and which consequently change with the parameter comprises the following process steps:
a) determining at least two different function values of measurement values, wherein each measurement value is assigned to a different energy, based on the same parameters,
b) determining at least two additional function values with the same energy correlation as in step a) based on at least two additional different parameters so that, for each energy value, there are at least three function values with three different parameters,
c) interpreting the function values belonging to the various energies as a parameter representation of an at least two-dimensional curve, and
d) evaluating the curve on the basis of predetermined criteria. The measurement values may be extinction values and the parameter may be time.
It is known that the separation and analysis processes of component mixtures operate, in their decisive phase, on the basis of registering those detection peaks, which are the result of spectroscopic measurement data. This is true for example for the HPLC-, the liquid-, the gas, the thin layer-, the affinity-, the adsorption-, and ion exchange chromatography, as well as for electrophoretic processes, and for any analog separation procedure.
From a simple and efficient elimination of the above-characterized central problem, an important technical advance for all automated separation and analysis processes can be expected.
The problems described earlier are eliminated by the process according to the invention. The process is direct, simple, free of delays and significant. It operates with a minimum of evaluation needs and leads to clear results.
The solution for the problem is centered about the detection peak.
Following the original classic separation processes, the number of analytical and preparative component separation procedures by way of chromatography has constantly increased. Alone in Europe, 100,000 such apparatus are in operation in all research and other professional areas. It can be assumed that this trend continues. Most detection and measurement methods in operation register the spectroscopic data dependent on the retention time and/or a travel behavior. The chromatograms obtained in this way are then used for the identification and the quantitative determination of individual components. It has been of utmost importance in these procedures that various components could be physically separated from each other to provide homogeneous individual signals.
With each chromatographic separation method on the basis of spectroscopic measurement methods, the basic question comes up again and again: How many components have a spectroscopic influence on the individual peaks of the chromatograms?
Because of the great importance of the individual contribution of each component to the more or less complex chromatogram, a large number of methods for the individual characterization of the components has been developed. The large efforts which have been expended in this respect and which are still being expended show that this problem has not been solved satisfactorily in spite of the continuing efforts.
A solution for this problem is of great importance for all measuring and separation methods, which are based on the registration of time-dependent signals. This importance applies also to any other method, wherein a signal appears and again disappears. Like in the chromatography, the individual separated bands can be examined spectroscopically in a time-dependent and position dependent manner also for example in a continuing electrophoresis process.
The drifting of basis lines and the partial disappearance of weak signals in noise are additional complication parameters for which substantial improvements are to be expected from the process according to the invention.
For concentrations at the detection limits, for example, at the sequencing limit of proteins the question is: Is it a real peak, a noise peak, an impurity peak, or a mixture of those?
A solution to these problems requires a new way of analyzing and treating detection peaks and their incorporation, in a reasonable manner, in a preferably fully automated over-all process.
For the peak analysis diode array, spectrometers are preferably used in the HPLC and the electrophoresis in the UV-VIS range. In this way, it is possible with today""s powerful computers to analyze chromatograms and electrograms spectrally down to the millisecond range. The 3D chromatograms and electrogams (extinction versus wavelength versus retention time or, respectively, an electrochemical parameter) registered in this way, are then generally subjected to a complicated numerical analysis. As examples, in this regard the following methods were developed.
Surface centerpoint analysis
peak deconvolution
vertical line and peeling method
spectra comparison using chemometric procedures
peak purity index
rationing (formation of ratios)
Most of these methods are complicated and expensive and are integrated in commercial apparatus which makes them very expensive. For example, multiple-variation methods or curve analyses on the basis of Gauss-distribution curves are utilized. All these methods however, have serious disadvantages:
The user can utilize these methods generally only schematically and routinely without any modification since these methods generally permit no changes as they are offered in the form of rigid uncontrollable xe2x80x9cblack-boxxe2x80x9d units, which cannot be changed.
The user cannot decide which results should be weighted more heavily when different procedures show contradicting results.
Numerical artifacts and problems with bad mathematical conditions are difficult to recognize during routine applications.
The experience of the user cannot be entered into the evaluation: the instrument cannot learn.
The extinction (E), extinction differences (ED), and extinction quotient (EDQ) diagrams were introduced in 1968 for the theory of the reaction kinetics. (H Mauser, Publication Naturforsch., 23b (1968), pages 1025-1030).
Herein, it was shown in connection with reacting chemical systems including some short-lived intermediates that, by plotting the timely subsequent extinction values at a wave length (xcexi) on the basis of the corresponding values at the same time at another wavelength (xcexj), while eliminating the time parameter, characteristic curves are obtained. Such diagrams Excexi versus Excexj are called extinction diagrams (=E diagrams).
If the differences between the extinction values are determined and plotted over the corresponding differences of other wave lengths, extinction difference diagrams (-ED- diagrams) are provided in the same manner. For forming the differences, extinction values at different reaction phases are utilized.
If corresponding differences of different wavelengths are divided by one another, extinction difference quotient diagrams (=EDQ-diagrams) are generated. Such differences represent determinants of the first grade. It is possible to continue with the system of determinants to the grades 2, 3 . . . to s.
Inspite of the existence of such diagrams in the theoretical field of kinetics since more than 25 years and the long-time efforts of the apparatus manufacturers to overcome the main weak point of peak interpretations, for example in the chromatography, the above diagrams have not been used so for rationalizing the processing of the flood of data supplied to the analysis apparatus. The reason herefor is probably that the kinetic examinations of reaction mechanisms and the chromatographic separation procedures for component mixtures have nothing in common. They serve completely different purposes. Reaction-kinetic theories and the practical problems of separation of compound mixtures are disciplines, which are widely separated. The introduction of E-diagrams into the separation of components was apparently not obvious.
It has now been found that, with the use of the E-, ED-, EDQ-diagrams, and particularly the integral extinction diagrams (=iE diagrams), which were specifically developed for the process according to the invention and which will be described below in connection with the analysis of the spectra, the substance coordination and determination in chromatographic separation processes becomes surprisingly extremely simple and comprehensible. It becomes instantly possible to determine whether there is a mono-peak, a double peak, or a multi-peak and the respective number of components involved, if applicable. Also, a noise peak included in the peak can be recognized. Complicated logistic auxiliary units become therefore superfluous. The total instrumentation is simplified, more reliable, and less expensive. Furthermore, the time required for obtaining an analysis result is substantially reduced.
The speed, the reliability, and the costs of the analysis are improved in a spectacular way.
In the chromatography, the detection method is based on the fact that the recorded spectra change dependent on the retention time. By plotting the extinctions of various wave lengths Excexi(t) resulting from the same retention times (t) with respect to each other, characteristic geometric figures are obtained which provide the desired characterization concerning the authenticity and the complexity of the signals examined. Such figures are points, straight lines, areas, or multidimensional shapes (polyhedra). Starting at a peak extremity having low E values, the curves evolve in the E diagram along such figures up to a reversal point with maximum E values and return to the original point in the E-diagram at the other peak extremity. The diagram formed in this way is called extinction space. The retention time generating the diagram is not contained therein any longer.
With the introduction of the simplified chromatographic analysis and separation process according to the invention a long-time need is satisfied. Purity, or respectively, multiplicity of the peaks is obtained in a most simple manner.
The invention may also be used in connection with any other process, wherein signals appear and disappear by means of timexe2x80x94or spacexe2x80x94evolving parameters (for example, NMR, absorptionxe2x80x94or reflexion spectroscopy of test carrier materials, double wave spectroscopy, fluorescence spectroscopy, optical rotation dispersion circular dichroism).
Before the development and an exemplary application of the various diagrams of the invention will be described, the capability is schematically shown in the following tables:
a) signal peaks with one or two components cover most of the chromatographic problems. They can be differentiated instantly using E- and EP diagrams.
b) The recognition and the treatment according to the invention of multi-component signals using further developed programs is possible.
Table 1 is expanded as a result according to the following scheme:
Embodiments of the invention will be described below on the basis of the accompanying drawings.